CN112813077B

CN112813077B - Application of Solyc01g007280 gene in resisting tomato yellow leaf curl virus

Info

Publication number: CN112813077B
Application number: CN202110294363.0A
Authority: CN
Inventors: 季晴宇; 赵统敏; 王银磊; 赵丽萍; 宋刘霞; 余文贵
Original assignee: Jiangsu University; Yangzhou University; Jiangsu Academy of Agricultural Sciences
Current assignee: Jiangsu University; Yangzhou University; Jiangsu Academy of Agricultural Sciences
Priority date: 2021-03-19
Filing date: 2021-03-19
Publication date: 2022-06-14
Anticipated expiration: 2041-03-19
Also published as: CN112813077A

Abstract

The invention relates to the technical field of crop genetic engineering, in particular to application of a Solyc01g007280 gene in resisting tomato yellow leaf curl virus, wherein the sequence of the Solyc01g007280 gene is shown as SEQ ID NO: 1 is shown. Solyc01g007280 gene is silenced by virus-induced gene silencing technology, and the gene-silenced plant can generate anti-TYLCV capability. The application of the invention is beneficial to the breeding of disease-resistant materials, can reduce the application amount of chemical pesticides in the production process, reduce the spread and prevalence of diseases, save the production cost, ensure the smooth development of tomato production and improve the economic benefit of the tomato industry.

Description

Application of Solyc01g007280 gene in resisting tomato yellow leaf curl virus

Technical Field

The invention relates to the technical field of crop genetic engineering, in particular to application of Solyc01g007280 genes in resisting tomato yellow leaf curl virus.

Background

The tomato is a annual or perennial plant of the genus Lycopersicon (Lycopersicon) of the family Solanaceae, and has high nutritive value. Native to south america, is one of the important vegetable crops in the world.

Tomato diseases are important factors for restricting the yield and quality of tomatoes, and Tomato Yellow Leaf Curl Virus (TYLCV) is an important virus threatening the development of Tomato industries around the world and is listed as one of ten important plant viruses in 2011. Tomato Yellow Leaf Curl Disease (TYLCD) was first discovered in Israel in 1931. In 1964, Israeli scientists Cohen and Harpaz found that the disease was transmitted by using Bemisia tabaci (Bemisia tabaci) as a vector for the first time, and named the disease tomato yellow leaf curl disease. Initially, the disease occurred only in the mediterranean generation, but with the increasing frequency of global warming, international transportation, tourism, trade and other human activities, bemisia tabaci, a vector, is prevalent worldwide, and tomato yellow leaf curl virus is spread worldwide.

At present, aiming at the prevention and treatment of tomato yellow leaf curl virus, the occurrence of diseases can be reduced by the traditional methods of physical prevention and treatment, chemical prevention and biological prevention and treatment, but the prevention and treatment are not thorough and are easy to repeat, and even harm can be caused to the environment. The virus belongs to Geminiviridae (Geminiviridae) single-stranded circular DNA virus, so that serious gene recombination exists, and tomato yellow leaf curl virus strains are more differentiated and have higher virus type variation, so that the disease is difficult to control, and once the tomato yellow leaf curl virus strains break out, serious harm is caused to tomato production. Meanwhile, the disease is mainly transmitted by the poison of the bemisia tabaci, so that the bemisia tabaci has strong reproductive capacity, wide host range and difficult control in production. At present, due to the mass propagation of the bemisia tabaci, certain difficulties exist in prevention and control, so the tomato breeding for disease resistance is still a main way for preventing and controlling the disease.

At present, the number of TYLCV genes identified from tomato materials is not large, and in addition, because fixed disease-resistant genes are used for a long time, pathogens are easy to mutate, so that the disease-resistant proteins cannot identify the invasion of the pathogens, and the resistance of the materials containing the disease-resistant genes is lost. Multiple virus co-infection may also result in loss of resistance of tomato to TYCLV.

Disclosure of Invention

The invention aims to provide the application of the Solyc01g007280 gene in resisting tomato yellow leaf curl virus, aiming at the problems that the existing anti-TYCLV material in tomato is insufficient and the disease-resistant material loses resistance in production.

In order to achieve the purpose, the invention provides the following technical scheme:

the early-stage research of the invention determines that the gene has extremely high resistance to TYLCV by introducing tomato materials containing the ty-5 gene and evaluating the resistance, and also determines that the disease-resistant gene is a single recessive genetic gene through genetic analysis, which is different from other tomato yellow leaf curl disease resistant genes controlled by known dominant genes. The allele Ty-5 of the resistance gene Ty-5 is a susceptible gene, and the loss of resistance of Ty-5 can endow susceptible material with resistance to TYCLV. The identification proves that the Moneymaker-Ty5 obtains excellent disease resistance to TYLCV by carrying out gene editing on Ty-5 in Moneymaker to create Ty-5 gene mutation and tomato material Moneymaker-Ty5 with 13 base deletions (see in particular China patent application CN201810735329.0, the content of which is incorporated herein in full and is not described in detail herein), and carrying out TYLCV inoculation on the material. Transcriptome sequencing is carried out before and after the inoculation of the material, and genes with differential expression are screened. Among the numerous genes, it was determined that Solyc01g007280 significantly upregulated expression following TYLCV infection. The VIGS technology is utilized to research the functions of the tomato gene, and the gene is determined to be a susceptible gene and can be used in the tomato TYLCV resistant breeding process.

According to the invention, TYLCV agrobacterium infectious clone inoculation is carried out on near isogenic system materials Moneymaker-Ty5 and Moneymaker, meanwhile, a sample inoculated with unloaded agrobacterium is used as a control to carry out sequencing on a transcriptome and a proteome, correlation analysis is carried out on the result, genes with obvious difference are screened, and Solyc01g007280 is determined as a candidate gene by combining GO analysis and KEGG analysis. After Virus-induced gene silencing (VIGS) technology is adopted, the gene-silenced plant after TYLCV inoculation is compared with a control to generate phenotype change, the phenotype change is determined to play a positive role in the TYLCV infection resisting process, the gene can generate the TYLCV resisting capability after silencing, and the gene is a disease-sensitive gene and can be used in the tomato TYLCV breeding resisting process.

The application of the Solyc01g007280 gene in resisting tomato yellow leaf curl virus diseases is disclosed, wherein the Solyc01g007280 gene sequence is shown as SEQ ID NO: 1 is shown.

The Solyc01g007280 gene is silenced by a virus-induced gene silencing technology, and a gene-silenced plant can generate the anti-TYLCV capability.

The application of the Solyc01g007280 gene in tomato TYLCV resistant breeding is provided, wherein the Solyc01g007280 gene sequence is shown as SEQ ID NO: 1 is shown.

Compared with the prior art, the invention has the beneficial effects that:

(1) aiming at the current situations that the current genes which can be used for tomato TYLCV breeding are lack and the resistance performance is poor, the invention digs out the gene Solyc01g007280 which can be used in the process of disease-resistant breeding, and the gene can be used as the supplement of other disease-resistant genes to improve the TYLCV resistance of tomatoes;

(2) the application of the invention is beneficial to the breeding of disease-resistant materials, can reduce the application amount of chemical pesticides in the production process, reduce the spread and prevalence of diseases, save the production cost, ensure the smooth development of tomato production and improve the economic benefit of the tomato industry.

Drawings

FIG. 1 is a schematic diagram of vector construction.

FIG. 2 shows the result of the target fragment amplification electrophoresis; wherein, M: marker; lane 1: high fidelity enzymatic amplification of the desired fragment.

FIG. 3 shows the result of detection by amplification electrophoresis; wherein, M: marker; lanes 1-4: amplifying the target fragment and the vector ligation product.

FIG. 4 shows the Solyc01g007280 silencing rate after tomato plants are inoculated with VIGS; wherein, CK: a control plant; 1-12: and (4) silencing the treated plants.

FIG. 5 is an analysis of disease index difference after tomato plants are inoculated with TYLCV; wherein, Solyc01g 007280: a gene-silenced plant; TRV2: control plants.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

First, experimental material

Plant material: the material Moneymaker for treating tomato yellow leaf curl;

the agrobacterium is infected with the attitude: GV 3101;

coli competence: DH5 a;

2 XT 5 Super PCR Mix (Colony), Taq DNA polymerase;

Loading buffer、DL2000 DNA Marker；

max DNA Polymerase high fidelity enzyme, SYBR Premix ExTaq quantitative kit, T4-DNA ligase, restriction enzymes BamH I and Xba I;

a total RNA extraction kit, a DNA purification and recovery kit, a high-purity plasmid small extraction kit and a DNA gel recovery kit;

HiScript II Q RT SuperMix for qPCR(+gDNA wiper)。

second, pTRV2 Solyc01g007280 vector construction

1. Primer design

VIGS primers (shown as SEQ ID NOS: 2-3) were designed using Primer 5, respectively, based on the known gene sequence:

VIGS primer F: 5'-GCTCTAGAACGCGTCACAGCAATTAGGA-3'

VIGS primer R: 5'-CGGGATCCCCCTCTCTTTCCGTACCTTCG-3'

Extracting Moneymaker plant total RNA and synthesizing cDNA; by using

Max DNA Polymerase high fidelity enzyme amplification target fragment, using restriction enzyme (BamH I and Xba I) to respectively perform double digestion 2% agarose gel cutting on vector pTRV2 plasmid and PCR product, recovering TRV2 vector, purifying PCR product; the target fragment was ligated with pTRV2 viral plasmid vector overnight at 16 ℃. As shown in fig. 1. Transforming the connected vector into escherichia coli competent DH5 alpha, plating overnight, and picking out the singleThe colonies were cultured overnight at 37 ℃ with shaking at 200 rpm. PCR amplification is carried out by taking the bacterial liquid as a template, sequencing analysis is carried out by detecting the amplification result through gel electrophoresis, and a sequencing primer is TRV2-F (shown as SEQ ID NO: 8): 5'-GGTCAAGGTACGTAGTAGAG-3' are provided. And extracting plasmids from bacteria with correct sequencing results, and transferring the plasmids into agrobacterium-infected GV3101 by an electric shock transformation method.

Third, VIGS inoculation

The pTRV2 strain Solyc01g007280 strain is subjected to plate cutting and shake culture, the strain is centrifugally collected, and MES infection solution is used for resuspending the strain to OD₆₀₀0.8-1, and adding 1: 1 pTRV1 was mixed with pTRV2: 00 and pTRV2: Solyc01g007280 respectively and left to stand for 3h, and Agrobacterium injection was carried out when the cotyledon of tomato plants (12 plants inoculated with both silent and control plants) was completely unfolded and no true leaf was grown.

Tetra, TYLCV inoculation

Tomato plants were grown with four leaves and one heart (about two weeks after inoculation of VIGS) in the same way (the staining solution was resuspended to OD)₆₀₀0.5) the TYLCV was inoculated directly and the severity index was counted weekly after inoculation. And carrying out gene silencing efficiency detection in the later period.

Five, Solyc01g007280 silencing

Semi-quantitative analysis of Solyc01g007280 silencing was performed by qRT-PCR with primers (shown in SEQ ID NO: 4-5):

Forward primer:5’-CGCGTCACAGCAATTAGG-3’

Reverse primer:5’-TGGAAAGAAAAGAATAGACAAGGA-3’

internal reference primers (shown as SEQ ID NO: 6-7):

Ubi-F:5’-TCGTAAGGAGTGCCCTAATGCTGA-3’

Ubi-R:5’-CAATCGCCTCCAGCCTTGTTGTAA-3’

and (4) analyzing results:

the length of the amplified target fragment is about 143bp, agarose electrophoresis detection is carried out on the product after PCR amplification, an electrophoresis band shows that a band with the size of 143bp is amplified (as shown in figure 2), the target band is connected with a TRV2 vector and is transformed by a TRV2-F (as shown in SEQ ID NO: 8): 5 ' -GGTCAAGGTACGTAGTAGAG-3 and Reverse primer (shown in SEQ ID NO: 5): 5'-TGGAAAGAAAAGAATAGACAAGGA-3' was PCR amplified (shown in FIG. 3) and subjected to sequencing analysis using TRV2-F to obtain a ligation-successful product, which was transformed into Agrobacterium and frozen at-80 ℃.

After the early stage inoculation of pTRV2: Solyc01g007280 and pTRV2: 00, TYLCV infection is carried out, and the disease index is investigated, and the silencing effect of the test is better according to the silencing rate detection result (as shown in figure 4). And the Solyc01g007280 silenced plants with enhanced resistance and the difference of disease index reaching the most significant level compared with the control plants can be obtained from the graphs in FIGS. 3 and 4 (FIG. 5).

Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that changes, modifications, substitutions and alterations can be made in these embodiments without departing from the principles and spirit of the invention, the scope of which is defined in the appended claims and their equivalents.

Sequence listing

<110> Yangzhou university

Jiangsu province academy of agricultural science

Jiangsu university

Application of <120> Solyc01g007280 gene in tomato yellow leaf curl virus disease resistance

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 970

<212> DNA

<213> Solyc01g007280(solanum lycopersicum)

<400> 1

atgtcgcgtt accgaggacc tcgtttcaaa aaaatacgac gcctgggggc tttaccagga 60

ctaactaata aaaagcctag gaccggaagt gatcttagaa accaatcgcg ttccggtaaa 120

aaatctcaat atcgtattcg tctagaagaa aaacaaaaat tgcgttttca ttatggtctt 180

acagaacgac aattacttaa atacgttcgt atcgccagaa aagccaaggg gtcaacaggt 240

caagttttac tacaattact tgaaatgcgt ttggataaca tcctttttcg attgggtatg 300

gcttccacta ttcccgcagc ccgtcaatta gttaaccatc gacatatttt agttaatggt 360

catatagtag atataccgag ttatcgctgc aaaccccgag atattattac ggcaaaggat 420

gaacaaaaat ccagagctct gattcaaatt tctctcgatt catcccctca tgaggaatta 480

ccaaatcatt tgacccttca gccattccaa tataaaggat tagtcaatca aataatagat 540

agtaaatggg tcggtttgaa aataaatgaa ttgctagtcg tagaatatta ttctcgtcag 600

acttaaacct cagacttaaa ccaagggttc gcaaaaattt cttcccttta ggcgaagtaa 660

attaacccaa ggttaagtaa gttaagtaag atcaatccga gtttgatcta gatttccccc 720

atttttgtat catagaccaa ggatctattt tcattccttg tctattcttt tctttccagt 780

attttgatta tgattatttt tgagtattct attttacgcg tcacagcaat taggaattga 840

gaatctatat ctttagatca aagaagggtc tagctgttgg aataatggta tacaatgcta 900

tttgagtcct tgttgttagt aaaattggtg aattaagcga aggtacggaa agagagggat 960

tcgaaccctc 970

<210> 2

<211> 28

<212> DNA

<213> VIGS primer F (artificial sequence)

<400> 2

gctctagaac gcgtcacagc aattagga 28

<210> 3

<211> 29

<212> DNA

<213> VIGS primer R (Artificial sequence)

<400> 3

cgggatcccc ctctctttcc gtaccttcg 29

<210> 4

<211> 18

<212> DNA

<213> primer F (Artificial sequence)

<400> 4

cgcgtcacag caattagg 18

<210> 5

<211> 24

<212> DNA

<213> primer R (Artificial sequence)

<400> 5

tggaaagaaa agaatagaca agga 24

<210> 6

<211> 24

<212> DNA

<213> Ubi-F(Artificial Sequence)

<400> 6

tcgtaaggag tgccctaatg ctga 24

<210> 7

<211> 24

<212> DNA

<213> Ubi-R(Artificial Sequence)

<400> 7

caatcgcctc cagccttgtt gtaa 24

<210> 8

<211> 20

<212> DNA

<213> TRV2-F(Artificial Sequence)

<400> 8

ggtcaaggta cgtagtagag 20

Claims

1. A method of enhancing resistance of tomatoes to yellow leaf curl virus, comprising: by silencingSolyc01g007280The gene enhances the resistance of the plant to TYLCV, wherein,Solyc01g007280the gene sequence is shown as SEQ ID NO: 1 is shown.

2. Method for enhancing the resistance of tomato to yellow leaf curl virus according to claim 1, characterized in that: by virus-induced gene silencing techniquesSolyc01g007280And (4) gene silencing.

3. A breeding method of tomato TYLCV is provided, wherein,Solyc01g007280the gene sequence is shown as SEQ ID NO: 1, is silent in tomato anti-TYLCV breedingSolyc01g007280A gene.